Lactose hydrolysis

Abstract

The invention features methods and compositions for rapidly and effectively hydrolyzing lactose using recombinant lactic acid bacteria.

What is claimed is:  
     
       1. A method for preparing a lactase microcarrier for hydrolyzing lactose in a liquid, the method comprising: 
       transforming a food-grade lactic acid bacterium with a DNA construct, wherein the DNA construct comprises a lantibiotic promoter sequence operatively linked to a DNA sequence encoding a β-galactosidase;  
       culturing the bacterium under conditions that enable expression of the β-galactosidase in an amount sufficient exhibit a β-galactosidase activity of at least 4000 Miller Units; and  
       permeabilizing the bacterium,  
       wherein a preparation of permeabilized bacteria equal to about 1.55 at OD 600  can hydrolyze either (i) about 100% of lactose in skim milk at a temperature of about 55° C. within two to three hours, (ii) about 50% of lactose in skim milk at a temperature of 4° C. within two to three hours, or (iii) both (i) and (ii).  
     
     
       2. The method of  claim 1 , wherein the lactic acid bacterium is selected from the group consisting of Streptococcus, Aerococcus, Carnobacterium, Enteroccus, Erysipelothrix, Gemella, Globicatella, Lactobacillus, Lactococcus, Bifodobacteria, Leuconostoc, Pediococcus, Tetragenococcus, and Bagococcus bacteria. 
     
     
       3. The method of  claim 1 , wherein the lactic acid bacterium is a  Lactococcus lactis.    
     
     
       4. The method of  claim 1 , wherein the DNA sequence encoding β-galactosidase is from a  Streptococcus thermophilus, Lactobacillus bulgaricus, Bifobacterium species, Aspergillus niger, Aspergillus oryzae, Kluyveromyces fragilis, Kluyveromyces lactis, Bacillus subtillus  or Arthrobacter species. 
     
     
       5. The method of  claim 1 , wherein the promoter is a nisin gene promoter. 
     
     
       6. The method of  claim 1 , wherein the promoter is a nisA promoter. 
     
     
       7. The method of  claim 1 , wherein the bacterium is permeabilized by an agent selected from the group consisting of a chemical, a solvent, and a detergent. 
     
     
       8. The method of  claim 1 , wherein the bacterium is permeabilized by ethanol, isopropanol, or a combination of ethanol and isopropanol. 
     
     
       9. The method of  claim 7 , wherein the detergent is selected from the group consisting of deoxycholate, sodium dodecyl sulfate, rhamnolipid, and chenodeoxycholate. 
     
     
       10. The method of  claim 1 , wherein the bacterium exhibits a β-galactosidase activity of at least 10,000 Miller Units. 
     
     
       11. A perneabilized lactic acid bacterium containing a heterologous β-galactosidase, wherein the bacterium exhibits a β-galactosidase activity of at least about 4000 Miller Units, and wherein a preparation of permeabilized bacteria equal to about 1.55 at OD 600  can hydrolyze either (i) about 100% of lactose in skim milk at a temperature of about 55° C. within two to three hours, (ii) about 50% of lactose in skim milk at a temperature of 4° C. within two to three hours, or (iii) both (ii and (ii). 
     
     
       12. The permeabilized bacterium of  claim 11 , wherein the bacterium is selected from the group consisting of Streptococcus, Aerococcus, Carnobacterium, Enteroccus, Erysipelothrix, Gemella, Globicatella, Lactobacillus, Lactococcus, Bifidobacteria, Leuconostoc, Pediococcus, Tetragenococcus, and Bagococcus bacteria. 
     
     
       13. The permeabilized bacterium of  claim 11 , wherein the bacterium is a  Lactococcus lactis.    
     
     
       14. The permeabilized bacterium of  claim 11 , wherein the β-galactosidase is a  Streptococcus thermophilus  β-galactosidase. 
     
     
       15. The permeabilized bacterium of  claim 11 , wherein the bacterium is in a lyophilized form, in a concentrated cell suspension, or immobilized. 
     
     
       16. A composition comprising the permeabilized bacterium of  claim 11 .